Breast cancer (BC) is the second cause of cancer death among women in the United States The development of brain metastases is qualified as the most advanced stage of the HER2+ expressing BC with survival rate from 3-6 months. Recently, a blocking antibody (Ab) to cytotoxic T lymphocyte (CTL)-associated antigen 4 (CTLA-4), which turns off the inhibitory mechanism to allow CTL and natural killer (NK) cells to eliminate cancer cells, was approved for cancer treatment. Working through regulatory T (Treg) cells enriched in tumors, this antibody dramatically activates anti-tumor immune response. An antibody blocking lymphocytic surface receptor programed cell death protein 1 (PD-1) has a similar activity. The immunotherapy of HER2+ BCs and its response to Treg inhibiting Abs (checkpoint inhibitors) is understudied and poorly understood, in spite of lymphocytic infiltration of their stroma. Our goal is to introduce new nano immunoconjugates (NIC) with attached checkpoint inhibitors that boost immune microenvironment of BCs and their brain metastases. Contrary to free checkpoint inhibitor Abs, our nano drugs will cross a number of bio barriers, including blood- brain barrier (BBB) and activate local primary breast and metastatic anti-tumor immune system. We propose to develop novel NICs loaded with the combination of tumor immune stimulators and inhibitors of cancer-specific marker(s), which is a response to the latest tendency in cancer treatment. As potent immune stimulators, we will use Abs to CTLA-4 or PD-1/PD-L1, which turn off the inhibitory mechanism allowing CTL to eliminate cancer cells. The same NIC will also have cytotoxic anti-cancer antisense drugs against (1) c-Myc, a master signaling regulator of cancer environment that can also suppress the expression of CTLA-4/PD-1, and/or (2) HER2 that would increase apoptosis of the tumor cells making them more visible to the immune system. A significant advantage of new NIC is the use of specific peptides to replace monoclonal Abs for the delivery to the tumor and across BBB. They will also make NIC less immunogenic and smaller in size. Three specific aims are proposed: Aim 1. Synthesize and characterize the nano immunodrugs; study the physico- chemical properties of poly(malic acid) (PMLA)-based NIC to optimize the synthesis with preservation of activity of peptide-based NIC delivery and treatment in comparison to delivery using tumor targeting Abs. Aim 2. Determine the efficacy of primary and metastatic HER2+ breast cancer treatment and mechanism of action of the nano immunodrugs. Analysis of T cell populations in BC to reveal reduced Treg (CD4+FOXP3+) fraction and increased CTL by nanopolymer conjugates and combinations. Aim 3. Define the pharmacokinetics and toxicity of the nano immunodrugs; study the pharmaceutical characteristics (half-life, PK), bio distribution, and toxicity of different NIC versions. The premedication against therapeutic Abs immuno toxicity will be used with anti-histamine triprolidine or/and PAF antagonist CV6209 to avoid acute immune response. Based on these studies we plan to select lead compound and administration strategy for further clinical development.